PROJECT SUMMARY The overall goal of this project is to understand the consequences of gene modification of hematopoietic stem progenitor cell (HSPC) by anti-HIV-1 transgenes which protect the harboring cells from HIV-1 infection or replication. We now know that stable long-term repopulation is a highly complex process whereby hundreds to thousands of HSPCs with differing life-spans and lineage output potentials contribute after transplant. We will investigate the behavior of human repopulating cells at the clonal level and use as our model, gene modification with anti-HIV-1 shRNA transgenes. We will test the hypothesis that in vivo selective pressures driven by HIV-1 or chemical means can expand clonal populations of gene-marked cells normally to reverse deficits caused by HIV-1. The earliest studies of hematopoietic stem cell transplant suggested that hematopoietic stem cells (HSC) were a relatively homogeneous population, any one of which has the potential to fully repopulate the hematopoietic system. However, more recently, this view has proven to be simplistic. Stem cell clones can be clustered into groups with widely differing kinetics of utilization and lineage commitment. One can consider repopulation by the stem cells as a process whereby hundreds or thousands of engrafted cells with differing properties compete in a highly controlled fashion to maintain homeostasis. It is generally thought that in vivo enrichment for the gene engineered HSPC or mature T-cells is critical in order to provide sufficient protected cells to withstand HIV-1 destruction. Studies have shown that HIV-1 infection imposes selection pressure for protected cells to survive in humanized mice when unprotected cells are killed by HIV-1. Further enrichment of gene-modified cells using chemoselection agents have been studied in animals. However, given that HSPC represent such diverse clonal populations, little is understood regarding the mechanism and degree to which HIV-1 and chemical agents exert selective pressure upon different HSPC clones. With the exception of the Berlin patient, where 100% of recipient cells were replaced by donor cells, no previous clinical studies established sufficient levels of protected T-cells to control HIV-1. Therefore, if we are to be successful in developing a stem cell-based therapy for HIV-1, we must fully investigate the extent and the mechanism by which HIV-1 and/or chemical agents can enrich for gene-modified cells after transplant of HSPC.